Induction of neutralizing antibodies against SARS-CoV-2 variants by a multivalent mRNA-lipid nanoparticle vaccine encoding SARS-CoV-2/SARS-CoV Spike protein receptor-binding domains in mice

To address the need for multivalent vaccines against Coronaviridae that can be rapidly developed and manufactured, we compared antibody responses against SARS-CoV, SARS-CoV-2, and several variants of concern in mice immunized with mRNA-lipid nanoparticle vaccines encoding homodimers or heterodimers of SARS-CoV/SARS-CoV-2 receptor-binding domains. All vaccine constructs induced robust anti-RBD antibody responses, and the heterodimeric vaccine elicited an IgG response capable of cross-neutralizing SARS-CoV, SARS-CoV-2 Wuhan-Hu-1, B.1.351 (beta), and B.1.617.2 (delta) variants.


Introduction
Beta-coronaviruses (beta-CoV) such as Middle East respiratory syndrome-associated coronavirus, severe acute respiratory syndrome coronavirus (SARS-CoV), and SARS-CoV-2, the causative agent of the recent COVID-19 pandemic started in early 2020 and was associated with high mortality rates worldwide [1,2].Experience with the Pfizer-BioNTech and Moderna mRNA-based SARS-CoV-2 vaccines approved in 2020/2021 underscores the importance of rapid deployment of vaccines for effective control measures [3,4].However, the emergence of more infectious and pathogenic variants of SARS-CoV-2 with enhanced immune escape has highlighted the need for multivalent vaccines that promote immunity to multiple SARS-CoV-2 variants for the current pandemic and, more broadly, to multiple members of the beta-CoV family.In this regard, mRNA vaccines have several advantages over protein-based or inactivated virus-based vaccines, including the feasibility of rapid design and synthesis, low-cost manufacture, and the availability of real-world clinical data supporting the safety of the mRNA platform in humans [5].Recently, a multivalent nucleoside-modified mRNA vaccine was developed that elicited high levels of cross-reactive and subtype specific antibodies against all known influenza virus subtypes [6].
The C-terminal receptor-binding domain (RBD) of the SARS-CoV-2 Spike glycoprotein interacts with angiotensin-converting enzyme 2 (ACE2), the human SARS-CoV-2 receptor, and thus plays a critical role in infection.Both the full-length surface Spike protein and the RBD are potent inducers of neutralizing antibodies and cellular immunity [7].However, the RBD is also the site of mutations in recently emerged SARS-CoV-2 variants of concern (VOC), including B.1.351(beta) B.1.617.2 (delta) [8] and omicron.Some of these mutations effectively reduce the neutralizing capacity of antibodies elicited by the current vaccines, resulting in increased transmissibility and/or pathogenicity [9,10].Therefore, development of multivalent vaccines must bear in mind the need for broad reactivity to diminish immune escape and protect against rapidly emerging variants [2-4, 7, 11-14].

Cell lines
HEK293FT and Vero E6 cells were maintained in Dulbecco's Modified Eagle's Medium containing 10% fetal bovine serum (GIBCO).The cell lines were tested and confirmed to be negative for mycoplasma.
The sequences were codon-optimized and cloned into pbluscript, an mRNA production plasmid generated in our lab.As shown in Supplementary Figure and Supplementary Data, the sequences comprised the T7 promoter, 5 0 and 3 0 untranslated regions of human hemoglobin subunit alpha 1, IgE signal peptide, and the respective RBD coding sequences.The DNA vectors were linearized and the mRNA was synthesized in vitro using T7 polymerase (Cellscript, #C-ASF3507), with UTP substituted by m1C-5'-triphosphate (TriLink, #N-1081).A donor methyl group S-adenosylmethionine was added to the methylated capped RNA (cap 0), resulting in a cap 1 structure to increase mRNA translation efficiency (Cellscript, #C-SCCS1710).The poly(A) tail was added using a Poly(A) Tailing Kit (Thermo Fisher Scientific, #74225Z25KU).

Verification of protein-coding capability of vaccine mRNAs
To confirm that the synthesized mRNAs could be translated into GFP or RBD proteins, 293FT cells were seeded at 3 × 10 5 cells/mL in 6-well plates, grown for 24 h, and then transfected with 1 mg mRNA per well using Lipofectamine 3000 (Invitrogen) according to the manufacturer's instructions.To prevent secretion of proteins, cells were incubated with the protein transport inhibitor brefeldin A (5.0 μg/mL) for 8 h after transfection.The transfected cells were cultured at 37˚C for 24 h and then collected and lysed using protein lysis buffer (Thermo Fisher Scientific, Cat #: 87787).Aliquots of lysate (20 μg protein) were resolved on 4-12% NuPAGE precast gels (Thermo Fisher Scientific) and transferred to PVDF membranes.RBD protein expression was analyzed using a rabbit polyclonal antibody SARS-CoV-2 Spike RBD Antibody (HRP) (Sino Biological, #40592-T62), which cross-reacts with SARS-CoV RBD protein.Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was probed as a loading control.

Immunization of mice with RBD mRNA-LNPs
This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.The protocol was approved by the Committee on the Ethics of Animal Experiments of the University of California San Diego (Protocol Number: S14123).All surgery was performed under ketamine anesthesia, and all efforts were made to minimize suffering.Male C57BL/6J mice (aged 4-5 weeks) were purchased from the Jackson Laboratory and housed according to the regulatory standards of the University of California, San Diego.Mice were randomly allocated to experimental groups.The mice were primed by intramuscular injection of mRNA-LNP (10 μg mRNA in 80 μL PBS) into the quadriceps muscle and then boosted 2 weeks later with the same mRNA-LNP dose and administration route.We divided the mRNA-LNP dose into 40 μl volume and injected intramuscularly into thigh muscle of both hindlimb of mice using 23-gauge needle.Blood was collected via cardiac puncture 2 weeks after the boost (4 weeks post-immunization) and serum was prepared and stored at −80˚C until analyzed.

SARS-CoV Spike protein-specific ELISAs
ELISAs were designed to quantify SARS-CoV-2 Spike protein-reactive total IgG, IgG2a, and IgG1, as well as SARS-CoV Spike protein-reactive total IgG.Recombinant SARS-CoV-2, SARS-CoV, or B.1.351Spike proteins (Sino Biological) were diluted to 200 ng/mL in 50 mM sodium carbonate buffer (pH 9.6), added to 96-well EIA/RIA plates (Corning) at 100 μL/well, and incubated overnight at 4˚C.The plates were washed with PBS containing 0.5% Tween-20 (PBST) and blocked with 5% bovine serum albumin (BSA) in PBS for 30 min at 37˚C.Mouse serum samples were serially diluted 5-fold in PBST containing 1% BSA and 100 μL was added to each well and incubated for 2 h at 37˚C.The plates were washed three times with PBST and incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (Abcam, ab6789, 1:10,000), goat anti-mouse IgG1 (Abcam, ab97240, 1:10,000), or goat anti-mouse IgG2a (Abcam, ab97245, 1:10,000) for 1 h.The plates were washed with PBST, color was developed by addition of 3 0 ,5,5 0 -tetramethylbenzidine (TMB) substrate, and the reaction was stopped by addition of 2 M HCl.The absorbance (optical density, OD) at 450 nm was measured using a microplate reader (BioTeK).The endpoint dilution titer was defined as the highest serum dilution giving an OD >2-fold the background OD of control wells consisting of diluted serum.Sera from human convalescent serum (HCS) was shown as a positive control, and sera from mouse immunized with GFP was shown as a negative control.

Neutralizing antibody assay with pseudoviruses
The neutralizing antibody titer of serum samples from vaccinated mice was determined by measuring the ability to block infection of Vero cells by chimeric VSVΔG-luc-SARS pseudoviruses (SARS-CoV, SARS-CoV-2 Wuhan-Hu-1, B.1.351,B.1.617.2,Wuhan-N501Y, or Wuhan-E484K).Vero cells were seeded at 2 × 10 5 cells/mL of 100 μL/well in 96-well plates and cultured overnight at 37˚C.Serum samples were heat inactivated at 56˚C for 30 min, serially diluted 3-fold in DMEM medium, mixed with VSVΔG-luc-SARS at 100 TCID 50 (50% tissue culture infectious dose), and incubated at 37˚C for 1 h.The mixture was then added to the plated Vero cells at 100 μL/well and incubated for 24 h at 37˚C.All samples were assayed in triplicate and controls consisting of Vero cells cultured alone or with pseudovirus without serum preincubation were tested in parallel.After 24 h, Vero cells were lysed and luciferase activity was measured using a Bright-Glo firefly luciferase kit (Promega).The 50% neutralization titer (NT 50 ) was calculated as the reciprocal of the highest serum dilution that gave a 50% reduction in luciferase signal compared with the negative control samples.NT 50 values were calculated using GraphPad Prism 8.0.

Statistical analysis
Data are presented as the mean and standard deviation, and symbols represent individual samples or mice.Group means were compared using ANOVA with Tukey's multiple comparison test (Fig 2 ), or a Mann-Whitney test (Fig 3).Statistical analyses were conducted using Graph-Pad Prism 8.0.A P value <0.05 was considered to be statistically significant.

Results and discussion
Recent work showed that homodimerization of beta-CoV RBDs increases their stability and immunogenicity [11,15], suggesting that homodimers or heterodimers of different beta-CoV RBDs might enhance their ability to elicit cross-reactive humoral and cellular immune responses.Therefore, in the present study, we designed and tested the immunogenicity of four candidate RBD mRNA-lipid nanoparticle (mRNA-LNP) vaccines in mice.We compared the vaccines' ability to elicit cross-reactive antibodies against not only the donor SARS-CoV and SARS-CoV-2 strains but also the recently emerged B.1.351and B.1.617.2VOC that exhibit enhanced immune evasiveness.
We designed five mRNA constructs encoding (1) green fluorescent protein (control), (2) SARS-CoV-2 Wuhan-Hu-1 reference strain RBD (R319-K537), (3) SARS-CoV CUHK-W1 RBD (R306-K523), (4) SARS-CoV-2 RBD homodimer, and (5) SARS-CoV/SARS-CoV-2 RBD heterodimer (Fig 1A).The mRNAs contained an N-terminal human IgE signal peptide to promote secretion; a modified nucleoside N1-methylpseudouridine to increase translation efficiency and reduce activation of the innate immune response [16]; and a cap1 modification at the end of the 5 0 -untranslated region (UTR) and a 3 0 -UTR poly(A) tail to increase stability and translation efficiency (S1 Fig) .Transfection of HEK293T cells with the RBD-encoding mRNAs resulted in high expression of each recombinant RBD, as determined by western blot analysis with a cross-reactive anti-SARS-CoV/SARS-CoV-2 RBD antibody (Fig 1B and S2 Fig), which confirmed the competency of the mRNAs to be translated in vivo.The RBD mRNAs were then mixed with a combination of lipids optimized to form lipid nanoparticles (LNPs), a commonly used simple and effective delivery vehicle for mRNA vaccines in vivo [17].Measurement of the diameters of multiple batches of RBD mRNA-LNPs by dynamic light scattering revealed minimal batch-to batch variation and an average particle diameter of 112 nm (Fig 1C ), a size that results in efficient tissue penetration and cellular uptake [18].
To evaluate the immunogenicity of the RBD mRNA-LNPs, groups of male C57BL/6 mice were immunized intramuscularly with 10 μg of each vaccine in 80 μL phosphate-buffered saline on days 0 and 14 (Fig 2A).Mice were bled 2 weeks after boosting and sera were analyzed for antibody production by direct binding ELISAs using SARS-CoV-2 and SARS-CoV purified Spike protein-coated plates.Notably, all four RBD mRNA-LNP vaccines elicited high titers of IgG reactive against both Spike proteins (Fig 2B and 2C).The mean titer of mice injected with As expected, the difference between these groups was not substantial, indicating that the specific immunogenicity of both monomers was similar.
To test if the breadth of the bivalent RBD was improved or reduced to SARS-CoV-2 VOCs, we included SARS_2-RBD monomeric (Group 2) and homomeric (Group 4) samples in the neutralizing assay against the Beta and Delta variants.The neutralizing titer against Beta was reduced by 5.5-, 5.3-, and 5. Taken together, these data identify a SARS-CoV/SARS-CoV-2 heterodimeric RBD mRNA-LNP vaccine candidate that has the capacity to elicit a strong SARS-CoV/SARS-CoV-2 cross-reactive binding and neutralizing antibody response in mice, including against several SARS-CoV-2 VOCs.One possible explanation on the increase of immunogenicity might be the induction of broad neutralizing antibodies.Further studies are needed to determine the effect of new antigens by combining RBD from multiple SARS variants to elicit cross-reactive antibodies.

Fig 1 .
Fig 1. Construction and characterization of multivalent RBD mRNA vaccines.a Schematic of the mRNA components of the vaccines.mRNAs encoded the signal peptide of human IgE followed by one or two copies of the receptor-binding domains (RBDs) of SARS-CoV-2 (Wuhan-Hu-1) or SARS-CoV (CUHK-W1) strains.A GFP-expressing vaccine was constructed as a control.b Western blot analysis of mRNA-encoded RBD protein expression.Each mRNA was in vitro transcribed and transfected into HEK293T cells for 24 h.Brefeldin A (5.0 μg/mL) was added to the cells at 8 h post-transfection to block protein secretion before cell lysis.Blots were probed with a rabbit anti-Spike antibody that recognizes both SARS-CoV and SARS-CoV-2 RBDs.GAPDH was probed as a loading control.c Distribution of RBD mRNA-LNP particle diameters measured by dynamic light scattering.For b and c, data from one experiment representative of three independent experiments are shown.https://doi.org/10.1371/journal.pone.0300524.g001